Methods for sequencing biopolymers
Inventors
Assignees
Arizona State University ASU • Arizona State University Downtown Phoenix campus
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Publication Number
US-12509720-B2
Publication Date
2025-12-30
Expiration Date
Abstract
The present disclosure provides devices, systems, and methods related to sequencing a biopolymer. In particular, the present disclosure relates to methods for sequencing a polynucleotide using a bioelectronic device that obtains a bioelectronic signature (e.g., current amplitude levels) of polymerase activity based on current fluctuations as complementary nucleotidepolyphosphate monomers (e.g., having distinct charges) are incorporated into the template polynucleotide.
Core Innovation
The invention relates to label-free polynucleotide sequencing using a bioelectronic device coupled to a polymerase. A template polynucleotide is provided, and distinctive-charge nucleotidepolyphosphate monomers are incorporated into the template. The bioelectronic device monitors polymerase activity through a bioelectronic signature including current fluctuations associated with open/closed state timing and current amplitude level differences.
The distinctive-charge nucleotidepolyphosphate monomers include distinctive negative charge conferred by additional phosphate groups and/or other charged moieties. Incorporation of each monomer/base is identified by correlations between the monomer distinctive charge and the observed polymerase current fluctuations. The document describes a distinctive-charge mechanism intended to differentiate incorporated nucleotidepolyphosphate monomers based on electrostatic charge effects on the polymerase current signature.
A bioelectronic device is also disclosed in which protein is attached to first and second electrodes via linkers at each attachment point. The linkers include distinctive negative electrical charge conferred by addition of at least one amino acid moiety, and the distinctive electrical charge modulates conductance through the protein.
The approach is described as extendable beyond polynucleotide sequencing by using other enzymes or protein-of-interest and by using RNA sequencing via RNA polymerases and rNTP analogs. The document connects the device-level charged-linker concept and the nucleotidepolyphosphate distinctive-charge monomer concept to monitoring enzyme activity with label-free bioelectronic signatures for determining sequence information.
Claims Coverage
The independent claim covers a bioelectronic device having a pair of electrodes separated by a gap, with a protein attached to both electrodes via charged linkers that confer a distinctive negative electrical charge; this charge modulates conductance through the protein. The dependent claims refine this concept by specifying the linker attachment chemistry, the nature of the negatively charged amino-acid moiety, the electrode materials, the selection of protein types, and functional constraints for polymerase activity.
Charged linker for conductance modulation
A protein is attached to the first and second electrodes via a linker at each attachment point comprising a distinctive negative electrical charge conferred by addition of at least one amino acid moiety, wherein the distinctive electrical charge modulates conductance through the protein.
Streptavidin/biotin attachment with distinctive negative charge
A linker comprising streptavidin and the protein being biotinylated such that the protein is attached to the electrodes via the streptavidin linker.
Amino-acid-derived distinctive negative charge moieties
The distinctive negative electrical charge is conferred by a linker incorporating a glutamate moiety, an aspartate moiety, or a combination thereof.
Selected electrode materials for device implementation
One or both of the first and/or second electrode comprises gold, palladium, platinum, silver, copper, or any alloys thereof.
Selected protein classes for the bioelectronic device
The protein is selected from polymerase, nuclease, proteasome, glycopeptidase, glycosidase, kinase, or endonuclease.
Polymerase with disabled exonuclease activity
The protein is a polymerase whose exonuclease activity is disabled.
Overall, the claim set centers on attaching a protein between two electrodes using a linker that includes a distinctive negative electrical charge from amino acid moieties, and using this charge to modulate conductance through the protein. Dependent claims specify streptavidin/biotin coupling, particular negative-charge amino acid moieties, electrode material selections, protein class selection, and disabling polymerase exonuclease activity.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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